Velvet based colony replication has been widely used for at least 50 years. Early velvet replicators were simple devices, no more than a round block used to support a sterile sheet of fabric via rubber band or hoop. In the early 1980""s Richard Provanchee of Philadelphia recognized some of the problems with traditional velvet replication and developed a replicator which employed a soft, compliant foam base to support the sterile transfer material which was not velvet but rather an open cell, flexible and water absorbent foam. This transfer surface was affixed to the flexible base via an adhesive.
In 1989 David Perlman developed a fabric-based replicator that did not employ a foam base but possessed so-called xe2x80x9cbumper guardsxe2x80x9d. These were apparently a slightly raised margin which allowed the device to accommodate the raised meniscus of agar plates. Perlman""s transfer surface was xe2x80x9cfixedly bondedxe2x80x9d to the replicator via an adhesive tab. He also included a marking element which indicated the orientation of replicated plates by indenting a portion of each plates agar surface.
Another problem with other replicators lies in the use of adhesives or rubber bands to affix the transfer materiel. Adhesives are messy and loose their xe2x80x9cStickxe2x80x9d over time. Rubber bands and loops can be difficult to position and must be passed over the sterile velvet surface without contacting and thereby contaminating it.
The transfer step is a time consuming bottleneck that severely limits the number of plates processed per day. To speed things up an old microbiological technique known as velvet plating may be employed. For this a cylindrical block of wood or plastic is placed on top of an inverted square of sterilized velvet. The four corners of overlapping velvet are next drawn up against the sides of the cylinder and a rubber band is drawn over these so as to hold the velvet in place. With the sterilized velvet in this way attached to the supporting block one can quickly lift all colonies from an incubated plate and transfer them to the surface of a new recipient plate or series of plates.
This sounds simple enough in theory, but in practice one soon finds that attempting to hold all four corners of velvet in position while trying to slip a rubber band over each while not touching and thereby contaminating the sterile velvet is easier said than done. Indeed, any touching of the velvet whatsoever may introduce contaminants. In addition to inconvenience and frustration other more serious problems inherent in the old method exist.
The first of these problems is caused by the peripheral wrinkles and overlapping foldings of fabric that can arise when one forces a square piece of loose cloth to conform to the form of a circle. This peripheral wrinkling causes what may be thought of as xe2x80x9ccontact shadowsxe2x80x9d. The piled up fabric of the wrinkle prevents adjacent, lower-lying velvet from contacting the agar. Thus the fabric in this shadow neither lifts colonies from the template nor transfers to the recipient plate. Although it is possible to prevent this problem via careful attention to detail while positioning the rubber band this takes added time, skill and again can increase the risk of surface contamination via excessive manipulation.
The second problem was a transfer failure of colonies. The root of this problem rests in the use of a flat, rigid cylinder base against which the velvet square is bound. If the surface of the Petri dish agar were as geometrically correct as the block base there would be no problem, however, due to the manner in which Petri plates are prepared this is not the case.
Agar media is prepared much like gelatin: a powder is added to water, it is dissolved, then autoclaved (heated at high temperature to sterilize) and poured into a mold. The difference is that in microbiology the mold is a flat and shallow Petri dish. As the added small volume of hot liquid agar is quickly swirled in order to fully cover the bottom of the plate it rapidly cools and solidifies. The result: the peripheral edge of agar in a Petri plate forms a raised lip against the wall of the plate.
When the replicator is subsequently pressed down on such a plate, the rigid velvet covered base presses down first on the higher agar of the peripheral lip region. Only with increased pressure is this agar lip region pressed down sufficiently so as to allow the centrally located colonies to be contacted by the sterile velvet This crushing of the peripheral agar to get at the central colonies can ruin any chance of getting a successful lift and transfer and often may destroy the template plate as well. This is because even if the crushed agar is not torn up as the velvet is removed, water forced from the agar matrix while under pressure, facilitates the mixing and cross-contamination of the minute bacterial cells which make up each colony.
Furthermore, this causes contamination of the replicator itself as the microscopic cells pass through the relatively loose grid of interwoven Velvet fibers. Replicator contamination makes It necessary to ethanol sterilize the device base prior to attachment of each new piece of velvet. Otherwise residual cells on the device can pass back through the fabric and cross-contaminate new plates.
The need for repeated sterilization of the device between each use is tedious and substantially adds to the time required to replicate multiple plates. Furthermore, one must ensure that all ethanol has evaporated since any residual disinfectant might kill the cells one is attempting to replicate: thereby leading to false negative results.
In a preferred embodiment of the present invention, a clonal replicator transfer disk is provided for transferring colonies from one agar surface to another. The transfer disk comprises a first side having a colony collecting substrate and a second side having an attachment means adapted to releasably engage a stamping base, the first side and the second side sandwiching a substantially fluid impervious layer.
In another preferred embodiment, the clonal reclicator transfer disk is substantially the shape of a Petri dish and the stamping base is contoured to mimic the shape of a prepared agar surface.
In yet another embodiment, the clonal replicator transfer disk comprises a marginal pull tab to assist in the removal of the transfer disk from the stamping base without the risk of contamination.
In another embodiment, the clonal replicator transfer is attached to the stamping pad with VELCRO.
In an additional embodiment, the stamping base further comprises an orientation marker to assist it proper orientation of the colonies on the agar surface.
In yet another embodiment of the clonal replicator transfer disk, the substantially fluid Impervious layer also exhibits resistant to high temperature to allow for autoclaving sterilization.
In an additional embodiment, the colony collecting substrate of the clonal replicator transfer disk is velvet.
In yet another embodiment, the colony collecting substrate of the clonal replicator transfer disk is suede.
The invention accordingly comprises the features of construction, combination of elements, and arrangement of parts that will be exemplified in the description set forth hereinafter and the scope of the invention will be indicated in the claims.
Other aspects and advantages of the present invention can be seen upon review of the figures, the detailed description, and the claims which follow.